The objectives of the project are to describe and define the operations of a DNA excision repair pathway in carcinogen-damaged human fibroblasts rendered permeable to enzymes, substrates and cofactors. Cells will be treated with 254 nm radiation or with (+-)-7alpha,8beta-dihydroxy-9beta,10beta-epoxy-7,8,9,10-tetrahydrobenzo(a)py rene to damage DNA, then permeabilized and incubated in vitro for assay of each step of excision repair. The conditions for assaying the strand incision step of repair in permeable cells will be optimized with attention directed to maximizing the signal of damage-directed strand incisions while minimizing non-specific DNA breakage. Reparative strand incisions, release of damaged DNA, gap-filling DNA synthesis and strand ligation all will be quantified in permeable cells and the efficiency of operation of each step compared with what is seen in intact cells. The sensitivity to digestion by micrococcal nuclease of repair patches synthesized in permeable cells will also be determined. The DNA repair assays will then be performed using permeable xeroderma pigmentosum cells with moderate or severe reparative deficiencies in order to quantify the reparative deficit in vitro. DNA-free human cell extracts or purified prokaryotic endonucleases will be added to deficient permeable cells to try to restore or stimulate reparative activity. Successful operation of repair in vitro will facilitate the characterization of enzymatic activities that are required of nucleotidyl DNA excision repair in human cells.